Compounds for the treatment of disorders related to vasculogenesis and/or angiogenesis

ABSTRACT

The present invention relates to organic molecules capable of modulating tyrosine kinase signal transduction and particularly KDR/FLK-1 receptor signal transduction in order to regulate and/or modulate vasculogenesis and angiogenesis. The invention is based, in part, on the demonstration that KDR/FLK-1 tyrosine kinase receptor expression is associated with endothelial cells and the identification of vascular endothelial growth factor (VEGF) as the high affinity ligand of FLK-1. These results indicate a major role for KDR/FLK-1 in the signaling system during vasculogenesis and angiogenesis. Engineering of host cells that express FLK-1 and the uses of expressed FLK-1 to evaluate and screen for drugs and analogs of VEGF involved in FLK-1 modulation by either agonist or antagonist activities is also described. 
     The invention also relates to the use of the disclosed compounds in the treatment of disorders, including cancer, diabetes, hemangioma and Kaposi&#39;s sarcoma, which are related to vasculogenesis and angiogenesis.

This application is a continuation-in-part of a U.S. application Ser.No. 08/386,021, filed Feb. 9, 1995, now U.S. Pat. No. 5,712,395, whichis a continuation-in-part of U.S. application Ser. No. 08/193,829, filedFeb. 9, 1994, which is a continuation-in-part of U.S. application Ser.No. 08/038,596, filed Mar. 26, 1993, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 07/975,750, filed Nov.13, 1992, now abandoned, all four of which are incorporated by referenceherein in their entirety.

1. Introduction

The present invention relates to novel compounds capable of modulatingand/or regulating tyrosine kinase signal transduction. The Applicantshave demonstrated that the murine fetal liver kinase 1 (FLK-1) receptorand its non-murine counterparts, including the human KinaseInsert-Domain-Containing Receptor (KDR), play a major role in a tyrosinekinase signal transduction system. Polypeptide growth factors such asvascular endothelial growth factor (VEGF) having a high affinity to theKDR/FLK-1 receptor have been associated with the proliferation ofendothelial cells and more particularly vasculogenesis and/orangiogenesis. Consequently, compounds affecting the enzymatic functionof the KDR/FLK-1 receptor may be used to not only regulate, modulateand/or inhibit the tyrosine kinase signal transduction system, but alsothe proliferation of endothelial cells in processes such asvasculogenesis and/or angiogeneis. The present invention is thereforefurther directed to the use of compounds which bind to and/or modulatethe activity of the receptors comprising the tyrosine signaltransduction system, and more specifically the KDR/FLK-1 receptor, totreat disorders related to vasculogenesis and/or angiogenesis.

2. Background of the Invention

Receptor Tyrosine Kinases. Receptor tyrosine kinases (RTKs) comprise alarge family of transmembrane receptors for polypeptide growth factorswith diverse biological activities. The intrinsic function of RTKs isactivated upon ligand binding, which results in phosphorylation of thereceptor and multiple cellular substrates, and subsequently in a varietyof cellular responses. Ullrich & Schlessinger, 1990, Cell 61:203-212.

As has been reported by the inventors, RTKs, as well as, more generally,protein tyrosine kinases, play an important role in the control of cellgrowth and differentiation (for review, see Schlessinger & Ullrich,1992, Neuron 9:383-391). Aberrant expression or mutations in the RTKshave been shown to lead to either uncontrolled cell proliferation (e.g.malignant tumor growth) or to defects in key developmental processes.Consequently, the biomedical community has expended significantresources to discover the specific biological role of members of the RTKfamily, their function in differentiation processes, their involvementin tumorigenesis and in other diseases, the biochemical mechanismsunderlying their signal transduction pathways activated upon ligandstimulation and the development of novel antineoplastic drugs.

At present, at least nineteen (19) distinct RTK subfamilies have beenidentified. One RTK subfamily is believed to be comprised of theKDR/FLK-1 receptor, the fetal liver kinase 4 (FLK-4) receptor and thefms-like tyrosine 1 (flt-1) receptor. Each of these receptors wasinitially believed to be receptors for hematopoietic growth factors.

The KDR/FLK-1 Receptor and VEGF. Normal vasculogenesis and angiogenesisplay important roles in a variety of physiological processes such asembryonic development, wound healing, organ regeneration and femalereproductive processes such as follicle development in the corpus luteumduring ovulation and placental growth after pregnancy. Folkman & Shing,1992, J. Biological Chem. 267(16):10931-34. Uncontrolled vasculogenesisand/or angiogenesis has been associated with diseases, such as diabetes,as well as malignant solid tumors that rely on vascularization forgrowth. Klagsburn & Soker, 1993, Current Biology 3(10):699-702; Folkham,1991, J. Natl., Cancer Inst. 82:4-6; Weidner, et al., 1991, New Engl. J.Med. 324:1-5.

Several polypeptides with in vitro endothelial cell growth promotingactivity have been identified. Examples include acidic and basicfibroblastic growth factor (FGF), vascular endothelial growth factor(VEGF) and placental growth factor. Unlike FGF, VEGF has recently beenreported to be an endothelial cell specific mitogen. Ferrara & Henzel,1989, Biochem. Biophys. Res. Comm. 161:851-858; Vaisman et al., 1990, J.Biol. Chem. 265:19461-19566.

Thus, identification of the specific receptors to which VEGF binds isimportant to understanding of the regulation of endothelial cellproliferation. Two structurally related RTKs have been identified tobind VEGF with high affinity: the flt-1 receptor (Shibuya et al., 1990,Oncogene 5:519-524; De Vries et al., 1992, Science 255:989-991) and theKDR/FLK-1 receptor, discussed herein. Consequently, it had been surmisedthat RTKs may have a role in the modulation and regulation ofendothelial cell proliferation.

As has only been recently contemplated, evidence, such as informationset forth in U.S. application Ser. Nos. 08/193,829, 08/038,596 and07/975,750, strongly suggest that VEGF is not only responsible forendothelial cell proliferation, but also is the prime regulator ofnormal and pathological angiogenesis. See generally, Klagsburn & Soker,1993, Current Biology 3(10)699-702; Houck, et al., 1992, J. Biol. Chem.267:26031-26037.

Identification Of Agonists And Antagonists To The KDR/FLK-1 Receptor. Inview of the surmised importance of RTKs to the control, regulation andmodulation of endothelial cell proliferation and potentiallyvasculogenesis and/or angiogenesis, many attempts have been made toidentify RTK "inhibitors" using a variety of approaches, including theuse of mutant ligands (U.S. application Ser. No. 4,966,849), solublereceptors and antibodies (Application No. WO 94/10202; Kendall & Thomas,1994, Proc. Nat'l Acad. Sci 90:10705-09; Kim, et al., 1993, Nature362:841-844), RNA ligands (Jellinek, et al., 1994, Biochemistry33:10450-56), protein kinase C inhibitors (Schuchter, et al., 1991,Cancer Res. 51:682-687); Takano, et al., 1993, Mol. Bio. Cell 4:358A;Kinsella, et al., 1992, Exp. Cell Res. 199:56-62; Wright, et al., 1992,J. Cellular Phys. 152:448-57) and tyrosine kinase inhibitors (WO94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No.5,330,992; Mariani, et al., 1994, Proc. Am. Assoc. Cancer Res. 35:2268).

More recently, attempts have been made to identify small molecules whichact as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclicor heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindolederivatives (PCT WO 94/14808) and 1-cycloproppyl-4-pyridyl-quinolones(U.S. Pat. No. 5,330,992) have been described generally as tyrosinekinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999),styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certainquinazoline derivatives (EP Application No. 0 566 266 Al), selenoindolesand selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCTWO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) havebeen described as compounds for use as tyrosine kinase inhibitors foruse in the treatment of cancer. None of these compounds, however, havebeen previously associated with the enzymatic function of the KDR/FLK-1receptor. Likewise, none of these compounds have been associated withregulation of vasculogenesis and/or angiogenesis.

The identification of effective small compounds which specificallyinhibit tyrosine signal transduction by modulating the activity of RTKsand particularly the KDR/FLK-1 receptor to regulate and modulatevasculogenesis and/or angiogenesis is therefore desirable and the objectof this invention.

3. Summary of the Invention

The present invention relates to organic molecules capable of modulatingtyrosine signal transduction and the use of such molecules to inhibit orpromote angiogenesis and/or vasculogenesis. Generally, the compounds ofthe instant invention are derivatives of quinazoline, quinoxaline,substituted aniline, isoxazoles, acrylonitrile and phenylacrylonitrilecompounds. More specifically, the invention is generally directed tocompounds having the formulae: ##STR1## and pharmaceutically acceptablesalts thereof, wherein:

R₁ is isopropyl, t-butyl, I, Br, OH or methyl,

R₂ is OH,

R₃ is 2-propyl, t-butyl, OH, H or methyl, and

R₄ is (1-phenyl)-n-propylaminocarbonyl, (E) 1-cyano-2-(3,5-diisopropyl-4-hydroxy)phenyl!ethenylsulfonyl, aminothiocarbonyl,cyanomethylsufonyl, (3-amino-4-cyano)pyrazo-5-yl,phenyl-n-propylaminocarbonyl, (E) 1-cyano-2-(5-bromo-3,4-dihydroxy)phenyl!ethenylsulfonyl,(1-phenyl)-n-propylaminothiocarbonyl, cyano, (E) 4-1-cyano-2(3,4-dihydroxy)phenyl!ethenyl!carbonylamino!-n-butyl!aminocarbonyl,benzylaminocarbonyl, 22-cyano-1-(3,4-dihydroxy)phenyl!ethylenyl!!sulfonyl,(3,4-dihydroxy)phenyl!carbonyl, (E) 4-1-cyano-2(3,4-dihydroxy)phenyl!ethenyl!carbonylamino!-ethyl!aminocarbonylor hydroxycarbonyl;

    --OR-- ##STR2## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is CH.sub.3 or H,

R₂ is CH₃ or H, or alternatively,

R₁ and R₂ form a phenyl ring (CHCHCHCH),

R₃ is H or formyl or chloro, and

R₄ is phenyl, (3,4-dihydroxy)phenyl, (4-iodophenyl)amino,(3,4-dichlorophenyl)amino, (3-chlorophenyl)amino, (4-bromophenyl)aminoor n-propylamino;

    --OR-- ##STR3## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is OCH.sub.3, CH.sub.3 or H,

R₂ is OCH₃ or H,

R₃ is H or chloro, and

R₄ is (3-chlorophenyl)amino, (4-methylphenyl)mecapto,(4-iodophenyl)amino or (3-hydroxyphenyl)amino;

    --OR-- ##STR4## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is 4-hydroxy-phenyl

R₂ is benzyl, and

R₃ is CH₃ ;

    --OR-- ##STR5## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is (3-trifluormethyl)phenyl and

R₂ is (2-chlorophenyl)aminothiocarbonyl;

    --OR-- ##STR6## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is O,

R₂ is (3,4-dihydroxyphenyl), and

R₃, R₄ and R₅ are H;

    --OR-- ##STR7## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is 4-(1-nitro)thiophene or indol-3-yl or indol-5-yl and

R₂ is aminothiocarbonyl, (3-amino-4-cyano)pyrazol-5-yl or(3,4-dihydroxyphenyl)carbonyl;

    --OR-- ##STR8## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is 2,5-dihydroxylbenzyl,

R₂ is H,

R₃ is methoxycarbonyl, and

R₄ is H;

    --OR-- ##STR9## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is H,

R₂ is (4-trifluoromethyl)phenyl, and

R₃ is methyl;

--OR-- ##STR10## and pharmaceutically acceptable salts thereof, whereinR₁ is (3-chloro)phenylamino.

The present invention is further directed to pharmaceutical compositionscomprising a pharmaceutically effective amount of the above-describedcompounds and a pharmaceutically acceptable carrier or excipient. Such acomposition is believed to affect the enzymatic activity of theKDR/FLK-1 receptor by, inter alia, inhibiting the signal transduced byinteraction between KDR/FLK-1 and vascular endothelial growth factor(VEGF) which may be useful in inhibition of diseases related tovasculogenesis and/or angiogenesis, including diabetes and cancer.Alternatively, such composition may act directly on the cellsresponsible for the disease (e.g. tumor cells). More particularly, thecompositions of the present invention may be included in methods fortreating, among other diseases, diabetic retinopathy, glioma, melanoma,Kaposi's sarcoma, hemangioma and ovarian, breast, lung, pancreatic,prostate, colon and epidermoid cancer. Such a composition, if used tomodulate, rather than inhibit, vasculogenesis and/or angiogenesis mayalso be useful in the promotion of wound healing.

Finally, the present invention is also directed to methods for treatingdiseases related to pathological vasculogenesis and/or angiogenesis,including but not limited to diabetes, diabetic retinopathy, rheumatoidarthritis, hemangioma and cancer and more particularly cancer related tosolid cell tumor growth (e.g., glioblastoma, melanoma and Kaposi'ssarcoma and ovarian, lung, mammary, prostate, pancreatic, colon andepidermoid carcinoma).

3.1. Definitions

"Pharmaceutically acceptable acid addition salt" refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are obtained by reaction with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

"Alkyl" refers to a saturated or unsaturated branched or straight chainhydrocarbon radical. Typical alkyl groups includes methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl andthe like.

4. Detailed Description of the Invention

The present invention relates to compounds capable of regulating and/ormodulating tyrosine signal transduction and more particularly KDR/FLK-1receptor signal transduction in order to inhibit or promote angiogenesisand/or vasculogenesis.

The present invention is based, in part, on the discovery that theKDR/FLK-1 tyrosine kinase receptor, and RTKs more generally, isexpressed on the surface of endothelial cells and may play a role inendothelial cell growth, including solid cell tumor growth. Theinvention is also based on the identification of VEGF as a high affinityligand of KDR/FLK-1 and the characterization of KDR/FLK-1 as an RTKrather than a hematopoietic receptor. Thus, the surmised role of VEGF inendothelial cell proliferation and migration during angiogenesis andvasculogenesis indicate an important role for the KDR/FLK-1 in theseprocesses.

The invention is further based on the observation that diseases such asdiabetes mellitus (Folkman, 1987, in XIth Congress of Thrombosis andHaemostasis (Verstraeta, et al., eds.) pp. 583-596, Leuven UniversityPress, Leuven) and arthritis, as well as malignant tumor growth mayresult from uncontrolled angiogenesis. See e.g., Folkman, 1971, N. Engl.J. Med. 285:1182-1186. Finally, the invention is based upon thediscovery and design of compounds that inhibit, prevent, or interferewith the signal transduced by KDR/FLK-1 when activated by ligands suchas VEGF. Although it is therefore believed that the compounds of thepresent invention act on a receptor or other component along thetyrosine kinase signal transduction pathway, the compounds may also actdirectly on the tumor cells that result from uncontrolled angiogenesis.

For purposes of this application, although the nomenclature of the humanand murine counterparts of the generic "flk-1" receptor differ, theyare, in many respects, interchangeable. The murine receptor, FLK-1, andits human counterpart, KDR, share a sequence homology of 93.4% withinthe intracellular domain. Likewise, murine FLK-1 binds human VEGF withthe same affinity as mouse VEGF, and accordingly, is activated by theligand derived from either species. Millauer et al., 1993, Cell72:835-846; Quinn et al., 1993, Proc. Natl. Acad. Sci. USA 90:7533-7537.FLK-1 also associates with and subsequently tyrosine phosphorylateshuman RTK substrates (e.g., PLC-γ or p85) when coexpressed in 293 cells(human embryonal kidney fibroblasts).

Models which rely upon the FLK-1 receptor therefore are directlyapplicable to understanding the KDR receptor. For example, use of themurine FLK-1 receptor in methods to identify compounds which regulatethe signal transduction pathway are directly applicable to theidentification of compounds which may be used to regulate the humansignal transduction pathway, and more specifically, activity related tothe KDR receptor. Angiogenesis is a very complex process involving theinvasion of endothelial cells into the nonvascularized tissue. No invitro model exists which mimics exactly this multistep processcomprising the degradation of the basal membrane surrounding theendothelial cells, migration into the perivascular stroma and eventuallyproliferation and formation of the new vascular sprout. Thus, chemicalcompounds identified as inhibitors of KDR/FLK-1 in vitro, will beconfirmed in suitable in vivo models. Both in vivo mouse and rat animalmodels have been demonstrated to be of excellent value for theexamination of the clinical potential of agents acting on the KDR/FLK-1induced signal transduction pathway.

In sum, the receptors to which VEGF specifically binds are an importantand powerful therapeutical target for the regulation and modulation ofvasculogenesis and/or angiogenesis and a variety of severe diseaseswhich involve abnormal cellular growth caused by such processes.Plowman, et al., 1994, DN&P 7(6):334-339. More particularly, theKDR/FLK-1 receptor's high specificity and role in the neovascularizationmake it a very distinct and powerful target for therapeutic approachesfor the treat cancer and other diseases which involve the uncontrolledformation of blood vessels.

This invention is therefore directed to compounds which regulate,modulate and/or inhibit vasculogenesis and/or angiogenesis by affectingthe enzymatic activity of the KDR/FLK-1 receptor and interfering withthe signal transduced by KDR/FLK-1. More particularly, the presentinvention is directed to compounds which regulate, modulate and/orinhibit the KDR/FLK-1 mediated signal transduction pathway as atherapeutic approach to cure many kinds of solid tumors, including butnot limited to glioblastoma, melanoma and Kaposi's sarcoma, and ovarian,lung, mammary, prostate, pancreatic, colon and epidermoid carcinoma. Inaddition, data suggest the administration of compounds which inhibit theKDR/FLK-1 mediated signal transduction pathway to the treatment ofhemangioma and diabetic retinopathy.

The invention also relates to the inhibition of vasculogenesis andangiogenesis via other receptor-mediated pathways, including the pathwaycomprising the highly related flt-1 receptor. Receptor tyrosine kinasemediated signal transduction is initiated by extracellular interactionwith a specific growth factor (ligand), followed by receptordimerization, transient stimulation of the intrinsic protein tyrosinekinase activity and autophosphorylation. Binding sites are therebycreated for intracellular signal transduction molecules and lead to theformation of complexes with a spectrum of cytoplasmic signallingmolecules that facilitate the appropriate cellular response. (E.g., celldivision, metabolic effects to the extracellular microenvironment) See,Schlessinger and Ullrich, 1992, Neuron 9:1-20.

It has been shown that tyrosine autophosphorylation sites in growthfactor receptors, such as KDR/FLK-1 and flt-1, function as high-affinitybinding sites for SH2 (src homology) domains of signaling molecules.Fantl et al., 1992, Cell 69:413-423; Songyang et al., 1994, Mol. Cell.Biol. 14:2777-2785); Songyang et al., 1993, Cell 72:767-778; and Koch etal., 1991, Science 252:668-678. Several intracellular substrate proteinsthat associate with receptor tyrosine kinases have been identified. Theymay be divided into two principal groups: (1) substrates which have acatalytic domain; and (2) substrates which lack such domain but serve asadapters and associate with catalytically active molecules. Songyang etal., 1993, Cell 72:767-778. The specificity of the interactions betweenreceptors, such as KDR/FLK-1 and flt-1, and SH2 domains of theirsubstrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. Theseobservations suggest that the function of each receptor tyrosine kinaseis determined not only by its pattern of expression and ligandavailability but also by the array of downstream signal transductionpathways that are activated by a particular receptor. Thus,autophosphorylation provides an important regulatory step whichdetermines the selectivity of signaling pathways recruited by specificgrowth factor receptors, as well as differentiation factor receptors.

The close homology of the intracellular regions of KDR/FLK-1 with thatof the PDGF-β-Receptor (50.3% homolgy) and/or the highly related flt-1receptor indicates the induction of overlapping signal transductionpathways. For example, for the PDGF-β-Receptor, members of the srcfamily (Twamley et al., 1993, Proc. Natl. Acad. Sci. USA 90:7696-7700),phosphatidylinositol-3'-kinase (Hu et al., 1992, Mol. Cell. Biol.12:981-990), phospholipase C-γ (Kashishian & Cooper, 1993, Mol. Cell.Biol. 4:49-51), ras-GTPase-activating protein, (Kashishian et al., 1992,EMBO J. 11:1373-1382), PTP-1D/syp (Kazlauskas et al., 1993, Proc. Natl.Acad. Sci. USA 90:6939-6943), Grb2 (Arvidsson et al., 1994, Mol. Cell.Biol. 14:6715-6726), and the adapter molecules Shc and Nck (Nishimura etal., 1993, Mol. Cell. Biol. 13:6889-6896), have been shown to bind toregions involving different autophosphorylation sites. For a generalreview, please see Claesson-Welsh, 1994, Prog. Growth Factor Res.5:37-54. Thus, it is likely that signal transduction pathways activatedby KDR/FLK-1 include the ras pathway (Rozakis et al., 1992, Nature360:689-692), the PI-3'-kinase pathway and the src-mediated andplcγ-mediated pathways. Each of these pathways may play a critical rolein the angiogenic and/or vasculogenic effect of KDR/FLK-1 in endothelialcells. Consequently, the present invention is also directed to the useof the organic compounds discussed herein to modulate angiogenesis andvasculogenesis as such processes are controlled by these pathways. 4.1.The Compounds

The invention is generally directed to compounds and/or compositionscomprising compounds having the formulae: ##STR11## and pharmaceuticallyacceptable salts thereof, wherein: R₁ is isopropyl, t-butyl, I, Br, OHor methyl,

R₂ is OH,

R₃ is 2-propyl, t-butyl, OH, H or methyl, and

R₄ is (1-phenyl)-n-propylaminocarbonyl, (E) 1-cyano-2-(3,5-diisopropyl-4-hydroxy)phenyl!ethenylsulfonyl, aminothiocarbonyl,cyanomethylsufonyl, (3-amino-4-cyano)pyrazo-5-yl,phenyl-n-propylaminocarbonyl, (E) 1-cyano-2-(5-bromo-3,4-dihydroxy)phenyl!ethenylsulfonyl,(1-phenyl)-n-propylaminothiocarbonyl, cyano, (E) 4-1-cyano-2(3,4-dihydroxy)phenyl!ethenyl!carbonylamino!-n-butyl!aminocarbonyl, benzylaminocarbonyl, 22-cyano-1-(3,4-dihydroxy)phenyl!ethylenyl!!sulfonyl,(3,4-dihydroxy)phenyl!carbonyl, (E) 4-1-cyano-2(3,4-dihydroxy)phenyl!ethenyl!carbonylamino!-ethyl!aminocarbonylor hydroxycarbonyl;

    --OR-- ##STR12## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is CH.sub.3 or H,

R₂ is CH₃ or H, or alternatively,

R₁ and R₂ form a phenyl ring (CHCHCHCH),

R₃ is H or formyl or chloro, and

R₄ is phenyl, (3,4-dihydroxy)phenyl, (4-iodophenyl)amino,(3,4-dichlorophenyl)amino, (3-chlorophenyl)amino, (4-bromophenyl)aminoor n-propylamino;

    --OR-- ##STR13## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is OCH.sub.3, CH.sub.3 or H,

R₂ is OCH₃ or H,

R₃ is H or chloro, and

R₄ is (3-chlorophenyl)amino, (4-methylphenyl)mecapto,(4-iodophenyl)amino or (3-hydroxyphenyl)amino;

    --OR-- ##STR14## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is 4-hydroxy-phenyl

R₂ is benzyl, and

R₃ is CH₃ ;

    --OR-- ##STR15## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is (3-trifluormethyl)phenyl and

R₂ is (2-chlorophenyl) aminothiocarbonyl;

    --OR-- ##STR16## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is O,

R₂ is (3,4-dihydroxy)phenyl, and

R₃, R₄ and R₅ are H;

    --OR-- ##STR17## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is 4-(l-nitro)thiophene or indol-3-yl or indol-5-yl and

R₂ is aminothiocarbonyl, (3-amino-4-cyano)pyrazol-5-yl or(3,4-dihydroxyphenyl)carbonyl;

    --OR-- ##STR18## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is 2,5-dihydroxybenzyl,

R₂ is H,

R₃ is methoxycarbonyl, and

R₄ is H;

    --OR-- ##STR19## and pharmaceutically acceptable salts thereof, wherein: R.sub.1 is H,

R₂ is (4-trifluoromethyl)phenyl, and

R₃ is methyl;

    --OR-- ##STR20## and pharmaceutically acceptable salts thereof, wherein R.sub.1 is (3-chloro)phenylamino.

The chemical formulae referred herein may exhibit the phenomenon oftautomerism. As the formulae drawings within this specification can onlyrepresent one of the possible tautomeric forms, it should be understoodthat the invention encompasses any tautomeric form which possesses theability to regulate and/or modulate vasculogenesis and/or angiogenesisand is not limited to any one tautomeric form utilized within theformulae drawings.

In addition to the above compounds and their pharmaceutically acceptablesalts, the invention is further directed, where applicable, to solvatedas well as unsolvated forms of the compounds (e.g. hydrated forms)having the ability to regulate and/or modulate vasculogenesis and/orangiogenesis.

The compounds described above may be prepared by any process known to beapplicable to the preparation of chemically-related compounds. Suitableprocesses are illustrated by the following representative examples.Necessary starting materials may be obtained by standard procedures oforganic chemistry.

4.2. Pharmaceutical Formulations And Routes Of Administration

The identified compounds can be administered to a human patient, byitself, or in pharmaceutical compositions where it is mixed withsuitable carriers or excipient(s) at doses to treat or ameliorate avariety of disorders, including solid cell tumor growth, includingKaposi's sarcoma, glioblastoma, and melanoma and ovarian, lung, mammary,prostate, pancreatic, colon and epidermoid carcinoma, diabetes, diabeticretinopathy, hemangioma and rheumatoid arthritis. A therapeuticallyeffective dose further refers to that amount of the compound sufficientto result in amelioration of symptoms of uncontrolled vasculogenesis andangiogenesis. Techniques for formulation and administration of thecompounds of the instant application may be found in "Remington'sPharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latestedition.

4.2.1. Routes Of Administration

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

4.2.2. Composition/Formulation

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration,the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1 with a5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80; the fraction size ofpolyethylene glycol may be varied; other biocompatible polymers mayreplace polyethylene glycol, e.g. polyvinyl pyrrolidone; and othersugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Many of the KDR/FLK-1 receptor modulating compounds of the invention maybe provided as salts with pharmaceutically compatible counterions.Pharmaceutically compatible salts may be formed with many acids,including but not limited to hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents that are the corresponding free base forms.4.2.3. Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC50 asdetermined in cell culture (i.e., the concentration of the test compoundwhich achieves a half-maximal inhibition of the RTK activity). Suchinformation can be used to more accurately determine useful doses inhumans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD50 and ED50. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See e.g. Finglet al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thekinase modulating effects, or minimal effective concentration (MEC). TheMEC will vary for each compound but can be estimated from in vitro data;e.g., the concentration necessary to achieve a 50-90% inhibition of thekinase using the assays described herein. Dosages necessary to achievethe MEC will depend on individual characteristics and route ofadministration. However, HPLC assays or bioassays can be used todetermine plasma concentrations.

Dosage intervals can also be determined using the MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

4.2.4. Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabelled for treatment of an indicated condition. Suitable conditionsindicated on the label may include treatment of a tumor, such as aglioma or glioblastoma and inhibition of angiogenesis.

5. Example

Compound Synthesis

5.1. Synthesis Of(E)-2-aminothiocarbonyl-3-(3,5-di-t-butyl-4-hydroxyphenyl)acrylonitrile

A preferred method of synthesis of (E)-2-aminothiocarbonyl-3(3,5-di-t-butyl-4-hydroxyphenyl)acrylonitrile (Compound 1) is asfollows: The compound was prepared as generally described by Ohmichi etal., 1993, Biochemistry 32:4650. A mixture of 0.47 g of3,5-Di-tert-butyl-4-hydroxybenzaldehyde, 0.2 g of thiocyanoacetamide and30 mg of β-alanine in 40 ml of ethanol was refluxed for 6 hours. Waterand HCl were added, and the reaction mixture was extracted with ethylacetate. Evaporation gave 0.34 g (54% yield) of a yellow solid having amelting point of 210° C.

The product gave the following analytical data: NMR (acetone-d₆) δ 8.47(1,H, s, vinyl), 8.02 (2 H, s), 1.48 (18 H, s); MS m/e 316 (M+, 100),303 (35), 301 (99), 268 (16), 260-(M (CH₃) 2=C,45), 245 (17), 228 (22),219 (52), 203 (10), 143 (11), 129 (11).

5.2. Synthesis Of (E)-2-cyano-3-(3-iodo-4,5-hydroxyphenyl)acrylonitrile

A preferred method of synthesis of(E)-1-cyano-3-(3-iodo-4,5-hydroxyphenyl)acrylonitrile (Compound 2) is asfollows: The compound was prepared as described in Ohmichi et al., 1993,Biochemistry 32:4650 in two steps. First,3-methoxy-4-hydroxy-5-iodobenzylidene malononitrile was prepared byadding 3 drops of piperidine to 1.4 g of 5-iodovanillin and 0.4 g ofmalononitrile in 25 ml of ethanol and refluxing the mixture for 4 hours.0.8 g (49% yield) of a yellow solid resulted.

The 3-methoxy-4-hydroxy-5-(iodobenzylidene)malononitrile product gavethe following analytical data: mp 188° C.; NMR (CDCl₃) δ 7.76 (1 H,J=1.8Hz, H6), 7.65 (1 H,d,J=1.8 Hz, H2), 7.56 (1 H,s,vinyl), 6.85 (1, H,s,OH), 3.99 (3, H,S, OCH₃); MS m/e 327 (13), 326 (M+, 100), 283 (18), 128(35), 101 (22).

Next, (3-methoxy-4-hydroxy-5-iodobenzylidene)malononitrile (0.65 g) and0.6 ml of boron tribromide (BBr₃) in 40 ml of dichloromethane werestirred under argon for 1 hour at room temperature. Water was added, andthe reaction mixture was extracted with ethyl acetate to give 0.46 g(73% yield) of a light-red solid (yellow in solution) having a meltingpoint of 105° C.

The final product gave the following analytical data: NMR (acetone-d₆) δ8.03 (1 H,s,vinyl), 7.88 (1 H,d,J=2.3 Hz, H2), 7.72 (1 H,d,J=2.3 Hz,H6); MS m/e 312 (M+, 38), 254 (74), 185 (M-I,27), 158 (M-I-HCN,11), 157(64), 130 (19), 129 (23), 127 (100).

5.3. Synthesis Of(E)-2-(3-phenyl-n-propylaminocarbonyl)-3-(3-bromo-4,5-diydroxyphenyl)acrylonitrile

A preferred method of synthesis of(E)-2-(3-phenyl-n-propylaminocarbonyl)-3-(3-bromo-4,5-diydroxyphenyl)acrylonitrile(Compound 3) is as follows:

1. A mixture of 0.69 g of 2.5 mM 5-iodovanillin, 0.5 g ofN-3-phenyl-n-propyl cyanoacetamide and 50 mg β-alanine in 30 ml ethanolwas refluxed for 5 hours. Evaporation gave an oil which was trituratedwith benzene-hexane and filtered to give 0.82 g of a bright yellow solid(71% yield) having a melting point of 83° C. Notably, the compound wasobserved to partially deteriorate over time when stored at room light.It is therefore preferred that the compound be stored as a solid andprotected from light. The product gave the following analytical data:NMR (CDCl₃) δ 8.12 (1H,S), 7.75 (1H,d,J=2.0 Hz), 7.68 (1H,d,J=2.0 Hz),7.30-7.10 (5H,m), 3.96 (3H,S,OCH₃), 3.45 (2H,q,J=6.0 Hz), 2.70 (2H, t,J=6.0 Hz), 1.95 (2H, quin, J=6.0 Hz). MS m/e 462 (M+,53), 357 (M-CH₂)₃Ph,18), 335 (M-I,100), 327(M-NH(CH₂)₃ Ph, 31).

2. 0.5 g of the compound of step 1 (3-methoxy-4-hydroxy-5-iodo α-ciscinnamone(3'phenylpropane)amide) and 0.4 ml of BBr₃ in 30 mldichloromethane were then stirred at room temperature for 1.5 hours.Water was added and the reaction extracted with ethyl acetate.Evaporation and trituration with benzene-hexane gave 0.3 g of a lightbrown solid (63% yield) having a melting temperature of 184° C.

The product gave the following analytical data: NMR (acetone d₆) δ 8.01(1H,S vinyl), 7.88 (1H,d,J=2.0 Hz), 7.66 (1H,d,J=2.0 Hz), 7.30(5H,m,Ph), 3.42 (2H,t,J=6.0 Hz), 2.70 (2H,t,J=6.0 Hz), 1.96 (2H, quin.,J=6.0 Hz). MS m/e 448 (M+, 3%), 321 (M-I,8), 217 (21), 201 (33), 118(100), m/e.

5.4. Synthesis Of (E)-2-(3-amino-4-cyano)pyrazo-5-yl!-3-(3,5-di-t-butyl-4-hydroxyphenyl)acrylonitrile

A preferred method of synthesis of (E)-2-(3-amino-4-cyano)pyrazo-5-yl!-3-(3,5-di-t-butyl-4-hydroxyphenyl)acrylonitrile(Compound 4) is as follows: A mixture of 0.7 g of3,5-di-t-butyl-4-hydroxybenzaldehyde, 0.46 g of3-amino-4-cyano-5-cyanomethyl pyrazole (prepared according to Carboni etal., 1958, J. Chem. Soc., 80:2838) and 40 mg of β-alanine was refluxedfor 15 hours. Cooling and filtering gave 0.5 g (46% yield) of yellowsolid having a melting point of 255° C.

The product gave the following analytical data: NMR (CDCl₃) δ 7.92(1H,S,vinyl), 7.80 (2H,S), 5.76 (1H,S,OH), 3.75 (2H,Br,S,NH₂), 1.48(18H,S). MS m/e 364(M+1,28), 363 (M+,100%), 348 (M-CH₃,58%), 292(M-56-CH₃,31%), 147 (41%), m/e.

5.5. Synthesis Of(E)-2-(3-phenyl-n-propylaminocarbonyl)-3-(3-isopropyl-4-hydroxy-5-(2-propylphenyl)acrylonitrile

A preferred method of synthesis of(E)-2-(3-phenyl-n-propylaminocarbonyl)-3-(3-isopropyl-4-hydroxy-5-(2-propylphenyl)acrylonitrile(Compound 5) is as follows: A mixture of 0.4 g of3,5-diisopropyl-6-hydroxy-benzaldehyde (for synthesis protocol, seeSection 5.11(step 2)), 0.55 g of N-3-phenyl-n-propyl cyanoacetamide(synthesized according to the protocol set forth in Gazit, et al., 1991,J. Med. Chem. 34(6):1896-1907) and 40 mg of β-alanine (in 20 ml ethanol)was refluxed for 5 hours. Workup (H₂ O, HCl, dichloromethane) gave anoil which crystallized on standing. Trituration with benzene-hexane gave0.4 g light yellow solid having a melting point of 120° C. Another 0.55g of solid was obtained from the from the mother liquid. The overallyield was 65%.

The product gave the following analytical data: NMR (CDCl₃) δ8.24(1H,S,vinyl), 7.73(2H,S), 7.23(5H,m), 5.50(1H,S,OH), 3.46(2H,q,J=6.7Hz, NH--CH₂), 3.17(2H, Septet, J=7.0 Hz), 2.71(2H,t,J=6.7 Hz), 1.95(2H,quintet, J=6.7 Hz), 1.30(12H,d,J=7.0 Hz).

5.6. Synthesis Of(E)-2-(benzylaminocarbonyl)-3-(3-iodo-4,5-dihydroxyphenyl)acrylonitrile

A preferred method of synthesis of(E)-2-(benzylaminocarbonyl)-3-(3-iodo-4,5-dihydroxyphenyl)acrylonitrile(Compound 6) is as follows: A mixture of 0.4 g of2-cyano-3-(4-hydroxy-3-iodo-5-methoxy)phenylacrylonitrile (which wasprepared by condensation of 4-hydroxy-3-iodo-5-methoxybenzaldehyde withN-benzylcyanoacetamide) and 0.5 ml of BBr₃ in 20 ml dichloromethane wasstirred for 2 hours at room temperature. Workup (H₂ O, ethyl acetate)gave 0.16 g of a yellow solid (41% yield) having a melting point of 220°C.

The product gave the following analytical data: NMR (acetone-d₆) δ8.05(1H,S,vinyl), 7.85(1H,d,J=2.1 Hz), 7.70(1H,d,J=2.1 Hz), 7.30(5H,m),4.6(2H,S).

5.7. Synthesis Of (E,E)-2- 2-1-cyano-2(3,4-dihydroxyphenyl)ethenyl!carbonylamino!ethyl!aminocarbonyl-3-(3,4-dihydroxyphenyl)acrylonitrile

A preferred method of synthesis of (E,E)-2- 2-1-cyano-2(3,4-dihydroxyphenyl)ethenyl!carbonylamino!ethyl!aminocarbonyl!-3-(3,4-dihydroxyphenyl)acrylonitrile (Compound 7) is as follows: Amixture of 0.7 g of 3.4-dihydroxy benzaldehyde, 0.5 g ofN-(cyanomethylcarbonylamino-n-propyl)cyanoacetamide and 4 dropspiperidine in 20 ml ethanol was refluxed for 4 hours. Water was added.Extraction with ethyl acetate, evaporation and trituration withethanol-dichloromethane gave 0.34 g (32% yield) of a yellow solid,having a melting point of 277° C.

5.8. Synthesis Of (E,E)-2- 4-1-cyano-2-(3,4-dihydroxyphenyl)ethenyl!carbonylamino!-n-butyl!aminocarbonyl!-3-(3,4-dihydroxyphenyl)acrylonitrile

A preferred method of synthesis of (E,E)-2- 4-1-cyano-2-(3,4-dihydroxyphenyl)ethenyl!carbonylamino!-n-butyl!aminocarbonyl!-3-(3,4-dihydroxyphenyl)acrylonitrile(Compound 8) is as follows: The compound was prepared according to theprotocol prescribed for the synthesis of compound above (Section 5.7,above). Following this protocol, a yellow solid (86% yield) resultedhaving a melting point of 283° C.

The product gave the following analytical data: NMR (acetone-d₆) δ 8.25(1H,5,vinyl), 7.23 (2H,5, H2,6).

5.9. Synthesis Of (E,E)- 2-2-cyano-2-(3,4-dihydroxyphenylcarbonyl)ethenyl!sulfonyl!-3-(3,4-dihydroxyphenyl)acrylonitrile

A preferred method of synthesis of (E,E)- 2-2-cyano-2-(3,4-dihydroxyphenylcarbonyl)ethenyl!sulfonyl!-3-(3,4-dihydroxyphenyl)acrylonitrile(Compound 9) is as follows: A mixture of 0.3 g ofN-(2-cyanomethylcarbonylaminoethyl)cyanoacetamide and 0.55 g of3,4-dihydroxy benzaldehyde with 30 mg β-alanine in 30 ml ethanol wasrefluxed for 6 hours and worked up. Evaporation of the ethyl acetate andtrituration with acetone-benzene gave a 0.45 g (58% yield) of a yellowsolid, having a melting point of 264° C. (stains hands).

The product gave the following analytical data: MS m/e 312 (28%), 265(24), 211 (100%), 185 (15%), 161 (44%), 160 (M-(compound structure),80%), 157 (35), 129 (22), 114 (43) m/e. NMR (acetone-d₆) δ 8.15(2H,s,vinyl), 7.77 (2H,d,J=2.3 Hz, H2), 7.59 (2H,dd,J=8.4, 2.3 Hz, H6),7.07 (2H,d,J=8.4 Hz, H5).

5.10. Synthesis Of (E)-3-(indol-5-yl)-2-(3,4-dihydroxyphenylcarbonyl)acrylonitrile

A preferred method of synthesis of(E)-3-(indol-5-yl)-2-(3,4-dihydroxyphenylcarbonyl)acrylonitrile(Compound 10) is as follows: A mixture of 130 mg of 5-formyl indole, 180mg of cyanomethyl-(3,4-dihydroxyphenyl)ketone (prepared according to theprotocol set forth at Gazit, et al., 1991, J. Med. Chem. 34:1896-1907)and 30 mg β-alanine was refluxed for 3 hours. Water was then added andthe reaction extracted with ethyl acetate to give an oily solidcontaining some aldehyde. Chromatography gave 86 mg of a pure orangesolid (28% yield) having a melting point of 185° C.

The product gave the following analytical data: MS m/e 304 (M+, 8%), 177(29), 137 (C₆ H₃ (OH)₂ CO⁺,100), 117 (12), 116 (indole+,15), 109 (93).NMR (acetone-d₆) δ 8.40 (1H,d,J=1.6 Hz, H₄), 8.18 (1H,S,vinyl), 8.03(1H,dd,J=8.6,1.6 Hz, H6), 7.63 (1H,d,J=8.6 Hz, H5), 7.54-7.40(3H,m,H3+H2,6), 7.0 (1H,d J=8.6 Hz, HS), 6.69 (1H, d J=3.2 Hz,H2).

5.11. Synthesis Of (E)-2-3-phenyl-n-propylaminothiocarbonyl!-3-(3,5-diisopropyl-4-hydroxyphenyl)acrylonitrile

A preferred method of synthesis of (E)-2-3-phenyl-n-propylaminothiocarbonyl!-3-(3,5-diisopropyl-4-hydroxyphenyl)acrylonitrile(Compound 11) is as follows:

1. A mixture of 6.2 g of N-phenylpropylcyanoacetamide (synthesizedaccording to the protocol set forth at Gazit, et al., 1991, J. Med.Chem. 34(6):1896-1907) and 15 g Lawsson reagent in 60 ml toluene wasrefluxed for 3 hours. Chromatography resulted in 1.5 g (22% yield) ofN-phenylpropylcyanothioacetamide as a red solid. The product gave thefollowing analytical data: NMR (CDCl₃) δ 7.3 (5H,m), 3.81 (2H,S), 3.71(2H,q, J=7.0 Hz), 2.74 (2H,t, J-7.0 Hz), 2.05 (2H, quintet, J-7.0 Hz).

2. A mixture of 18 g of 2,6-diisopropyl phenol and 1.8 g ofhexamethylenetriamine (HMTA) in 60 ml trifluoroacetic acid (TFA) wasrefluxed for 3.5 hours. Workup, chromatography and trituration withhexane gave 5.3 g (26% yield) of 3,8-diisopropyl-6-hydroxybenzaldehydewhite solid, having a melting point of 103° C. Analytical analysis ofthe product gave the following data: NMR (CDCl₃) δ 9.87(1H,S,CHO),7.63(2H,S), 3.19(2H,septet,J=7.7 Hz), 1.30(12H,d,J=7.7 Hz).

3. 0.6 g of the compound of Step 1 (N-phenylpropyl cyanothioacetamide),0.6 g of the compound of Step 2 and 40 mg β-alanine in 40 ml ethanolwere refluxed for 4 hours. Evaporation and chromatography gave 0.6 g(50% yield) of Compound 11 as a viscous oil. NMR (CDCl₃) δ8.76(1H,S,vinyl), 7.78(2H,S,H2.6), 7.25(5H,m), 5.60(1H,S,OH),3.90(2H,q,J=7.0 Hz), 3.17(2H,Septet, J=7.0 Hz), 2.76(2H,t,J=7.0 Hz),2.11(2H, quintet, J=7.0 Hz), 1.29(12H,d,J=7.0 Hz). MS m/e 407(M+1,55),406(M+,70), 373(M--CH₃ --H₂ O,100), 363(M-isopropyl, 72),272(M--NH(CH₂)₃ Ph, 20), 259 (58), 230 (28), 91 (28).

5.12. Synthesis Of (E)-2-1-cyano-2-(5-bromo-3,4-dihydroxyphenyl)ethenylsulfonyl!-3-(3-bromo-4,5-dihydroxyphenyl)acrylonitrile

A preferred method of synthesis of (E)-2-1-cyano-2-(5-bromo-3,4-dihydroxyphenyl)ethenylsulfonyl!-3-(3-bromo-4,5-dihydroxyphenyl)acrylonitrile (Compound 12) is as follows: 10 ml of ethanolcontaining 230 mg of 5-bromo 3.4-dihydroxy benzaledehyde, 76 mg ofdiacetonitrile sulphone and 10 mg of β-alanine were refluxed for 5hours. Cooling and filtering gave 220 mg (76% yield) of an orange solidhaving a melting point greater that 300° C.

The product gave the following analytical data: NMR (acetone-d₆) δ8.18(2H,S,vinyl), 7.90 (2H,d,J=1.6 Hz), 7.78 (2H,d,J=1.6 Hz).

5.13. Synthesis Of 2- (4-iodophenyl)amino!-6,7-dimethyl quinoxaline

A preferred method of synthesis of 2- (4-iodophenyl)amino!-6,7-dimethylquinoxaline (Compound 13) is as follows:

1. A mixture of 2 g of 4,5-dimethyl-1,2-diaminobenzene and 1.5 g ofglyoxylic acid hydrate in 30 ml ethanol was refluxed for 2 hours.Cooling and filtering gave 1.2 g (46% yield) of a white solid, having amelting point of 263° C. The product gave the following analytical data:

NMR (DMSO d₆) δ 60:40 mixture.

major--8.07 (1H,S), 7.55(1H,S), 7.06(1H,S), 2.30(6H,S).

minor--8.02 (1H,S), 7.42(1H,S), 7.28(1H,S), 2.28(6H,S).

2. A mixture of 1.1 g of the product of step 1(6,7-dimethylquinoxalone), 1 ml phosphrous oxychloride (POCl₃) and 1 mldimethyl aniline in 20 ml toluene were refluxed for 2 hours. Workup(NH₃, dichloromethane) and chromatography gave 0.4 g (33% yield) of awhite solid (2-chloro-6,7-dimethylquinoxaline), having a melting point86° C. The product gave the following analytical data: NMR (CDCl₃) δ8.68(1H,S,H₂), 7.85(1H,S), 7.76 (1H,S), 2.50(6H,S).

3. A mixture of 210 mg of the product of step 2(2-chloro-6,7-dimethylquinoxaline) and 0.8 g of p-iodoaniline was heatedin 10 ml ethanol at 100° C. for 4 hours. Chromatography gave 245 mg (60%yield) of a light green solid having a melting point of 228° C.

The product gave the following analytical data: NMR(CDCl₃) δ 8.32(1H,S),7.67(1H,S), 7.64(1H,S), 7.68, 7.56(4H,Abq, JAB=9.0 Hz).

5.14. Synthesis Of 2-(3,4-dihydroxyphenyl)-6,7-dimethylquinoxaline

A method of synthesis of 2- (3, 4-dihydroxyphenyl) -6,7-dimethylquinoxaline (Compound 14) is as follows: 1.4 g of 4.5-dimethyl1,2-phenylenediamine and 1.9 g of α-chloro 3,4-dihydroxy acetophenone in15 ml dimethylsulfoxide were heated for 1.5 hours at 100° C. 80 ml of H₂O was added and the suspension was left overnight at room temperatureand filtered to give 2.5 g (67% yield) of a brown solid.

The product gave the following analytical data: NMR (acetone-d₆) δ9.28(1H,S,H2), 8.40(Br.S,OH), 7.89(1H,d,J=2.2 Hz, H2'), 7.82(2H,S,H55,8), 7.72(1H,dd,J=8.3,2.2 Hz,H6'), 7.02 (1H,d,J=8.3 Hz,H5'),2.52 (6H,S,CH₃). DMSO d6 9.30(1H,S,H2), 7.81(2H,S,H5,8), 7.75(1H,d,J=2.2 Hz,H₂ '), 7.62(1H,dd,J=8.3,2.2 Hz,H6'), 6.90 (1H,d,J=8.3 Hz,H5'), 2.44(6H,S,CH₃).

A second method of synthesis of2-(3,4-dihydroxy)phenyl-6,7-dimethyl-quinoxaline is as follows: 1 g and1.9 g of the above reagents in 25 ml ethanol were refluxed 2 hours.Cooling and filtering gave 0.76 g (18% yield) of a deep yellow solidhaving a melting point of 278° C. as the HCl salt.

5.15. Synthesis Of 4-(4-iodophenylamino)-6,7-dimethoxy quinazoline

A preferred method of synthesis of the compound (Compound 15) is asfollows:

1. A mixture of 7 g of 4,5-dimethoxy 2-aminobenzoic acid and 8 ml offormamide was heated for 2 hours at 170° C. Cold water was added and thesolid filtered to give 0.9 g (12% yield) of a light-brown solid(6,7-dimethoxyquinazolone), having a melting point of 308° C.

The product gave the following analytical data: NMR(DMSO-d₆) δ8.0(1H,S), 7.43(1H,S), 7.12(1H,S), 3.89 (3H, S) , 3.85 (3H, S).

2. 0.8 g of the compound of Step 1. 1 ml POCl₃ and 1 ml dimethylanilinein 20 ml toluene were refluxed for 3.5 hours. Workup and triturationwith hexane gave 0.5 g of a light grey solid (57% yield), having amelting point of 188° C.

The product gave the following analytical data: NMR (CDCl₃) δ8.88(1H,S), 7.41(1H,S), 7.36(1H,S), 4.09(3H,S), 4.08(3H,S).

3. A mixture of 300 mg of 4-chloro-6,7-dimethoxyquinazoline and 300 mgof 3-iodoaniline in 10 ml ethanol was refluxed for 1 hour. Cooling andfiltering gave 540 mg (93% yield) of a white solid as the HCl salt. Thesolid had a melting point of 278° C.

The product gave the following analytical data: NMR (DMSO-d₆) δ8.87(1H,S,H2), 8.27(1H,S), 8.13(1H,S), 7.8-7.66(2H,m), 7.33(2H,m),4.02(3H,S), 4.0(3H,S).

5.16. Synthesis Of 4-(3-hydroxyphenylamino)-6-methylquinazoline

A preferred method of synthesis of4-(3-hydroxyphenylamino)-6-methylquinazoline (Compound 16) is asfollows:

1. A mixture of 0.8 g of 5-methyl-2-aminobenzoic acid and 15 mlformamide was heated at 170° C. for 1.5 hours. Water was added and thesolid filtered to give 7.3 g (83% yield) of a brown-white solid(6-methylquinazolone) having a melting point of 268° C.

2. A mixture of 5 g of the compound of step 1, 5 ml of POCl₃ and 5 mldimethyl aniline in 40 ml toluene were refluxed for 3.5 hours. Workup(NH₃, H₂ O, ethyl acetate) gave a dark solid. Chromatography yielded(29% yield) 1.61 g of a white solid (4-chloro-6-methyl-quinazoline)having a melting point of 98° C. The product gave the followinganalytical data: NMR (CDCl₃) δ 9.0 (1H,S,H2), 8.04(1H,d,J=2.0 HzH₅),7.96(1H,d,J=8.8 HzH₈), 7.80(1H,dd,J=8.8,2.0 Hz, H₇), 2.62(3H,S).

3. A mixture of 230 mg of the compound of step 2 and 145 mg ofm-aminophenol in 10 ml ethanol was refluxed for 50 minutes. Cooling andfiltering gave 300 mg (80% yield) of a light yellow solid, as the HClsalt. The product had a melting point of 262° C.

The product gave the following analytical data: (DMSO-d₆) δ8.89(1H,S,H2), 8.72 (1H,S,H5), 7.90(2H,ABq,J=8.0 Hz, H7,8), 7.2(3H,m),6.75(1H,m), 2.55(3,H,S).

5.17. Synthesis Of 2-(3,4-dichlorophenylamino)-6,7-dimethylquinoxaline

A preferred method of synthesis of2-(3,4-dichlorophenylamino)-6,7-dimethylquinoxaline (Compound 17) is asfollows: A mixture of 150 mg of the compound of2-chloro-6,7-dimethylquinoxaline (which may be synthesized according tothe protocol at Section 5.13 (step 2)) and 0.7 g of 3,4-dichloroanilinewas heated at 100° C. for 3.5 hours. Chromatography gave 80 mg (33%yield) of a yellow brown solid, having a melting point of 229° C.

The product gave the following analytical data: NMR (CDCl₃) δ8.32(1H,S), 8.16(1H,d,J=2.4 Hz,H2'), 7.71(1H,Br.S), 7.65(1H,Br.S),7.57(1H,dd,J=2.4, 9.2 Hz, H6'), 7.43(1H,d,J=9.2 Hz, H5'), 2.48(3H,S),2.46(3H,S).

5.18. Synthesis Of 4-(3-hydroxyphenylamino)quinazoline

A preferred method of synthesis of 4-(3-hydroxyphenylamino)-quinazoline(Compound 18) is as follows:

1. A mixture of 4.6 g of quinazolone, 5 ml of POCl₃ and 5 ml ofdimethylaniline in 50 ml toluene was refluxed for 3.5. hours. Workup(NH₃, H₂ O and ethylacetate) yielded a green oil. Chromotographyresulted in a light brown solid. Sublimation at 160° C. (11 mm Hg) gave1.48 g (29w yield) of a white solid (4-chloro-quinazoline) having amelting point of 72° C. This product gave the following analytical data:NMR (CDCl₃) δ 9.05 (1H,S), 8.27 (1H,m), 8.1-7.9 (2H,m) 7.75 (1H,m).

2. A mixture of 0.37 g of the compound of step 1 and 0.24 g ofm-hydroxyaniline in 10 ml ethanol was refluxed for 1 hour. Cooling andfiltering gave 0.25 g (41% yield) of a light-yellow solid as the HClsalt. The solid turns light green on standing overnight. The product hasmelting point of 268° C.

The product gave the following analytical data: NMR (DMSO-d₆) δ 8.99(1H,S), 8.93(2H,S), 8.15-7.81 (3H,m), 7.31-7.12(3H,m), 6.77(1H,d,J=7.4Hz).

5.19. Synthesis Of 2-(n-propylamino)-3-chloroquinoxaline

A preferred method of synthesis of 2-(n-propylamino)-3-chloroquinoxaline(Compound 19) is as follows: 0.8 g of 2-phenyl-3,6,7-trimethylquinoxaline 10 ml dimethylsulfoxide was heated for 40 minutes at 90° C.Workup and chromatography gave 40 mg (4% yield) of a white solid havinga melting point of 148° C. The product gave the following analyticaldata: NMR (CDCl₃) δ 8.03(1H,S), 7.90(1H,S), 7.60(5H,m), 6.96(1H,S,CHBr₂), 2.52(6H,S).

Following the same protocol, 0.3 g (47% yield) of a white solid wasobtained having a melting point of 150° C. The product gave thefollowing analytical data: NMR(CDCl₃) δ 11.30(1H,S,CHO), 8.05(1H,S),7.96(1H,S), 7.67(2H,m), 7.55(3H,m), 2.55(6H,5).

5.20. Synthesis Of 4- (4-methylphenyl)mercapto!quinazoline

A preferred method of synthesis of the compound (Compound 20) is asfollows: A mixture of 250 mg of 4-chloro-6-methyl-quinazoline, 180 mg ofp-thiocresole and 100 mg of potassium hydroxide (KOH) are combined in 20ml CH₃ CN and stirred 24 hours at room temperature. Workup (H₂ O,dichloromethane) and trituration with hexane gave 40 mg (10% yield) of alight blue solid having a melting point of 96° C.

The product gave the following analytical results: NMR (CDCl3) δ8.81(1H,S), 7.96(1H,d,J=2.0 Hz,H5), 7.85(1H,d,J=9.0 Hz, H8),7.68(1H,dd,J=9.0, 2.0 Hz, H7), 7.51, 7.30(4H,ABq,JAB=8.2 Hz),2.56(3H,S), 2.42(3H,S). MS m/e 266 (M+,40%), 265 (M-1,100%) m/e.

5.21. Synthesis Of2-chloro-4-(3-chlorophenylamino)-6,7-dimethoxyquinazoline

A preferred method of synthesis of2-chloro-4-(3-chlorophenylamino)-6,7-dimethoxyquinazoline (Compound 21)is as follows:

1. A mixture of 8 g of 6,7-dimethoxy-2,4-quinazolinedione, 23 ml ofPOCl₃ and 10 ml of dimethylaniline in 30 ml toluene was refluxed for 5hours. Workup (H₂ O, NH₃, dichloromethane) and titration with hexanegave 7 g (75% yield) of a light green solid(2,4-dichloro-6,7-dimethyoxy-quinazoline having a melting temperature of156° C. The product gave the following analytical results: NMR (CDCl₃) δ7.36(1H,S), 7.28(1H,S), 4.07(3H,S), 4.06(3H,S).

2. A mixture of 2.6 g of the compound of step 1 and 1.3 g ofm-chloroaniline in 20 ml ethanol was refluxed for 1 hour. The mixturewas then cooled and filtered to give 3.5 g (90% yield) pink-white solid.

Alternatively, a second method of synthesis using the free base wasused. 3.4 g of the material form above was treated with NH₃ --H₂ O andextracted with ethyl acetate. Recrystallization from benzene-hexane gave2.3 g (74% yield) of a white solid having a melting point of 222° C.

The product gave the following analytical data: NMR (CDCl₃) δ7.74(1H,t,J=2.2 Hz,H2'), 7.63(1H,m), 7.30(1H,m), 7.16(1H,S), 7.12(1H,m),6.98(1H,t,J=8.0 Hz), 4.0(3H,S), 3.97(3H,S).

5.22. Synthesis Of (Z)-1-(2-chlorophenyl)-2-2,2-dicyanoethenyl!hydrazine

A preferred method of synthesis of (Z)-1-(2-chlorophenyl)-2-2,2-dicyanoethenyl!hydrazine (Compound 22) is as follows: 2.4 g ofsodium nitrite (NaNO₂) was added to 4 g of m-chloro-aniline in 20 ml ofdiluted hydrochloric acid and 20 ml H₂ O and then cooled in ice forapproximately 0.5 hours. The mixture was then added into a solution of2.2 g malononitrile and 10 g potassium acetate in 100 ml ethanol. After0.5 hours in the cold and 1 hour at room temperature the solid wasfiltered, washed with water and dried to give 2.4 g (37% yield) yellowsolid.

The product gave the following analytical data: mp-170° C. NMR (CDCl₃) δ7.4-7.2, m.

5.23. Synthesis Of 2-phenyl-1,4-diaza-anthracene

A preferred method of synthesis of 2-phenyl-1,4-diaza-anthracene(Compound 23) is as follows: 20 ml of ethanol containing 0.47 grams of2,3-diaminonaphthalene and 0.47 grams of phenyl glyoxal hydrate wererefluxed for 1.5 hour. Cooling and filtering gave 0.5 g (65%) of a lightbrown solid with a melting point of 163° C.

The product gave the following analytical data: NMR (CDCl₃) : δ 9.38(1H, l.c., H2), 8.71, 8.67 (2H, 2d, H5,10), 8.25, 8.10 (4H, AA'BB'm,H6-9), 7.58(5H, m, Ph). MS m/e 256 (M+, 100%), 229 (M--CN, 12%),126(71%) m/e.

5.24. Synthesis Of N-(2,5-dihydroxylbenzyl)-4-methoxycarbonyl aniline

A preferred method of synthesis ofN-(2,5-dihydroxylbenzyl)-4-methoxycarbonylaniline (Compound 24) is asfollows: 0.7 g of 2,5-dihydroxybenzaldehyde and 0.75 g of3-aminomethylbenzoate in 40 ml of methanol were refluxed for 3 hours andcooled. 0.5 g of sodiumcyanoborohydride (NaCNBH₄) was then added. After12 hours at room temperature, workup (H₂ O, ethylacetate), andchromatography (silica gel, elution with 5% CH₃ OH in dichloromethane),0.42 g (31% yield) of a light yellow solid was obtained.

The product gave the following analytical data: mp 175° C. NMR(acetone-d₆) δ 7.78, 6.68 (4H, ABq, JAB=8.8 Hz), 6.74 (1H,d,J=3.0 Hz,H6), 6.72 (1H,d,J=8.5 Hz,H3), 6.55 (1H,d,J=8.5,3.0 Hz,H₄), 4.34(2H,s,CH₂ N), 3.76 (3H,s,COOCH₃).

5.25. Synthesis OfN-(2-chlorophenyl)-2-cyano-2-(N-3-trifluorophenylaminocarbonyl)-thioacetamide

N-(2-chlorophenyl)-2-cyano-2-(N-3-trifluorophenylamino-carbonyl)-thioacetamide(Compound 25) may be synthesized as follows:

680 mg of sodium ethoxide was added to a solution of 1.6 gram ofN-3-trifluoromethylphenyl cyanoacetamide in 20 ml of tetrahydrofuran at0° C. This mixture was stirred at 0° C. for 1 hour and 1.7 g of2-chlorophenylisothiocyanate in 5 ml of tetrahydrofuran was addeddropwise. After addition, the mixture was warmed to room temperature andheated at 50° C. for 6 hours. Upon cooling, all the ethanol was removedand the resulting solid was resuspended in 10 ml of water, This was thenadded to 15 ml of 0.3M sodium hydroxide solution, shaken vigorously andwashed in 50 ml of ethyl ether. The aqueous layer was then acidifiedwith 1N hydrochloric acid to pH 1. The solid was then collected bysuction filtration to produce 630 mg ofN-2-chlorophenyl(2-cyano-2-N-3-trifluorophenylaminocarbonyl)thioacetamide.

5.26. Synthesis Of(E)-2-cyanomethylsufonyl-3-(3-bromo-4,5-dihydroxyphenyl)acrylonitrile

A preferred method of synthesis of(E)-2-cyanomethylsufonyl-3-(3-bromo-4,5-dihydroxyphenyl)acrylonitrile(Compound 26) is as follows: A mixture of 500 mg of5-bromo,3,4-dihydroxybenzaldehyde and 700 mg of sulfonyldiacetonitrilein 6 ml of ethanol was refluxed with a few drops of piperidine for 4hours. Ethanol was removed in a rotavap and the mixture worked up withethyl acetate, diluted acid and brine. A portion of the crude was thenpurified by HPLC on a C-18 column to provide about 50 mg of(E)-2-cyanomethylsufonyl-3-(3-bromo-4,5-dihydroxyphenyl)acrylonitrile.

5.27. Synthesis Of (1-benzyl-2-hydroxyphenyl)pyrolido3,4-b!3,4-dihydro-4-oxoquinazoline

A preferred method of synthesis of (1-benzyl-2-hydroxyphenyl)pyrolido3,4-b!3,4-dihydro-4-oxoquinazoline (Compound 27) is as follows: 0.01 mol(1.07 g) of benzylamine and 0.01 mol (1.22 g) of 4-hydroxybenzaldehydewere mixed together in 15 ml of ethanol and refluxed on a waterbath for15 minutes. 0.01 mol (3.44 g) of 2-(1'-tosyloxyethyl)-quinazolin-4-oneand one drop of pyridine were added and the mixture was refluxed for sixhours. The resulting solution was evaporated and extracted with a 5%water solution of sodium bicarbonate. The remaining crystals werefiltered off, washed with water and recrystallized from isopropanol.3.40 g of compound was obtained (89% yield) having a melting point of217°-219° C.

The product (C₂₄ H₂₁ N₃ O₂) gave the following analytical data:

Elemental analysis %! of Product

Calculated: C: 75.18 H: 5.52 N: 10.96

Found: C: 75.26 H: 5.47 N: 10.88

5.28. Synthesis Of 2-(3-chlorophenylamino)-6,7-dimethylquinoxaline

A preferred method of synthesis of2-(3-chlorophenylamino)-6,7-dimethylquinoxaline (Compound 28) is asfollows: 200 mg of 2-chloro-6,7-dimethylquinoxaline and 700 mg m-chloroaniline were heated without solvent at 100° C. for 2.5 hours.Chromatography gave 100 mg of a white solid (34% yield) having a meltingpoint of 175° C.

The product gave the following data: NMR (CDCl₃) δ 8.33(1H,S),7.97(1H,m), 7.68(1H,S), 7.62(1H,S), 7.54(1H,m), 7.27(1H,m), 7.05(1H,m),2.45(3H,S), 2.43(3H,S). MS m/e 285, 283(M+,29,81%), 248(m-Cl,7).

5.29. Synthesis Of(E)-2-(3,4-dihydroxybenzoyl)-3-dihydroxyphenyl)acrylonitrile.

A preferred method of synthesis of(E)-2-(3,4-dihydroxybenzoyl)-3-dihydroxyphenyl)acrylonitrile (Compound29) is as follows: Solid potassium cyanide (KCN) (3 g, 46 mmol) wasadded to 2-chloro-3',4'-dihydroxyacetophenone (6 g) in 30 ml ofdimethylsulfoxide. The reaction mixture was stirred at 100° C. for 2.5hours. After cooling, 100 ml of 1N hydrochloric acid was added, and thereaction mixture was extracted with ethyl acetate. Drying andevaporation gave a red oily solid which was purified by chromatographyon silica gel. The first fractions eluted with 3% methanol indichloromethane were evaporated and triturated with dichloromethane.Filtering gave 1 g (18% yield) of a light yellow solid having a meltingpoint of 217° C.

The product gave the following analytical data: NMR (acetone-d6) δ7.51(2H,m,H2,6), 6.97 (1H,d,J=8.0 Hz, H5), 4.43 (2H,s,CH₂ CN).

5.30. Synthesis Of 2-(4-bromophenylamino)-6,7-dimethyl-quinoxaline

A preferred method of synthesis of2-(4-bromophenylamino)-6,7-dimethylquinoxaline (Compound 30) is asfollows: 200 mg of 2-chloro-6,7-dimethyl quinoxaline (synthesizedaccording to the protocol at Section 5.13, step 2) and 0.8 g ofp-bromoaniline were heated at 100° for 3.5 hours. Chromatography gave 25mg (37% yield) of a light yellow solid, having a melting point of 235°C.

The product gave the following analytical data: NMR (CDCl₃) δ8.32(1H,S), 7.68(1H,S), 7.60(1H,Br.S), 7.68, 7.48(4H,ABq, JAB=8.8 Hz),2.45(3H,S), 2.43(3H,S).

6. Example

ELISA Assay To Measure Kinase Activity Of FLK-1 Receptor In FLK-1/NIHCells

An ELISA assay was conducted to measure the kinase activity of the FLK-1receptor and more specifically, the inhibition or activiation of proteintyrosine kinase activity on the FLK-1 receptor.

6.1. Materials And Methods

Materials. The following reagents and supplies were used:

a. Corning 96-well ELISA plates (Corning Catalog No. 25805-96);

b. Cappel Goat anti-rabbit IgG (catalog no. 55641);

c. PBS (Gibco Catalog No. 450-1300EB);

d. TBSW Buffer (50 mM Tris (pH 7.2)m 150 mM NaCl and 0.1% Tween-20);

e. Ethanolamine stock (10% ethanolamine (pH 7.0), stored at 4° C.);

f. HNTG buffer (20mM HEPES buffer (pH 7.5), 150 mM NaCl, 0.2% TritonX-100, and 10% Glycerol);

g. EDTA (0.5M (pH 7.0) as a 100X stock);

h. Sodium Ortho Vanadate (0.5M as a 100X stock);

i. Sodium pyro phosphate (0.2M as a 100X stock);

j. NUNC 96 well V bottom polypropylene plates (Applied ScientificCatalog No. AS-72092);

k. NIH3T3C7#3 Cells (FLK-1 infected cells);

l. DMEM with 1X high glucose L Gulatamine (catalog No. 11965-050);

m. FBS, Gibco (catalog no. 16000-028);

n. L-glutamine, Gibco (catalog no. 25030-016);

o. VEGF, PeproTech, Inc. (catalog no. 100-20) (kept as 1 ug/100 ul stockin Milli-Q dH₂ O and stored at -20° C.;

p. Affinity purified anti-flk-1 antiserum, Enzymology Lab, Sugen, Inc.;

q. UB40 monoclonal antibody specific for phophotyrosine, Enzymology Lab,Sugen, Inc.;

r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog no. 172-1011);

s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) solution(100 mM citric acid (anhydrous), 250 mM Na₂ HPO₄ (pH 4.0), 0.5 mg/mlABTS (Sigma catalog no. A-1888)), solution should be stored in dark at4° C. until ready for use;

t. H₂ O₂ (30% solution) (Fisher catalog no. H325);

u. ABTS/H₂ O₂ (15 ml ABTS solution, 2 ul H₂ O₂) prepared 5 minutesbefore use and left at room temperature;

v. 0.2M HCl stock in H₂ O;

w. dimethylsulfoxide (100%)(Sigma Catalog No. D-8418); and

y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).

Protocol. The following protocol was used to conduct the ELISA Assay:

1. Coat Corning 96-well elisa plates with 1.0 ug per well CappelAnti-rabbit IgG antibody in 0.1M Na2CO3 pH 9.6. Bring final volume to150 ul per well. Coat plates overnight at 4° C. Plates can be kept up totwo weeks when stored at 4° C.

2. Grow cells in 30 ml of Growth media 9DMEM. 2.0 mM L-Glutamine, 10%FBS) until confluent in 150 cm tissue culture dishes at 37° C., 5% CO₂.

3. Harvest cells by tyrpsination and seed in Corning 25850 polystyrene96-well roundbottom cell plates, 25.000 cells/well in 200 uL of growthmedia.

4. Grow cells at least one day at 37° C., 5% CO₂.

5. Wash cells with D-PBS 1X.

6. Add 200 ul/well of starvation media (DMEM, 2.0 mM l-Glutamine, 0.1%FBS). Incubate overnight at 37° C., 5% CO₂.

7. Dilute Compounds/Extracts 1:20 in polyproplyene 96 well plates usingstarvation media. Dilute dimethylsulfoxide 1:20 for use in controlwells.

8. Remove starvation media from 96 well cell culture plates and add 162ul of fresh starvation media to each well.

9. Add 18 ul of 1:20 diluted Compound/Extract dilution (from step #7) toeach well plus the 1:20 dimethylsulfoxide dilution to the control wells(+/-VEGF), for a final dilution of 1:200 after cell stimulation. Finaldimethylsulfoxide is 0.5%. Incubate the plate at 37° C., 5% CO₂ for twohours.

10. Remove unbound antibody from Elisa plates by inverting plate toremove liquid. Wash 3 times with TBSW+0.5% Ethanolamine, pH 7.0. Pat theplate on a paper towel to remove excess liquid and bubbles.

11. Block plates with TBSW+0.5% Ethanolamine, pH 7.0. 150 ul per well.Incubate plate thirty minutes while shaking on a microtiter plateshaker.

12. Wash plate 3 times as described in step 10.

13. Add 0.5 ug/well affinity purified anti-flk-1 polyclonal rabbitantiserum. Bring final volume to 150 ul/well with TBSW+0.5k EthanolaminepH 7.0. Incubate plate for thirty minutes while shaking.

14. Add 180 μl starvation medium to the cells and stimulate cells with20 ul/well 10.0 mM Sodium Ortho Vanadate and 500 ng/ml VEGF (resultingin a final concentration of 1.0 mM Sodium Ortho Vanadate and 50 ng/mlVEGF per well) for eight minutes at 37° C., 5% CO₂. Negative controlwells receive only starvation medium.

15. After eight minutes, media are removed from the cells and washed onetime with 200 ul/well PBS.

16. Lyse cells in 150 ul/well HNTG while shaking at room temperature forfive minutes. HNTG formulation includes sodium ortho vanadate, sodiumpyro phosphate and EDTA.

17. Wash Elisa plate three times as described in step 10.

18. Transfer cell lysates from the cell plate to elisa plate andincubate while shaking for two hours. To transfer cell lysate pipette upand down while scrapping the wells.

19. Wash plate three times as described in step 10.

20. Incubate Elisa plate with 0.02 ug/well UB40 in TBSW+05%ethanolamine. Bring final volume to 150 ul/well. Incubate while shakingfor 30 minutes.

21. Wash plate three times as described in step 10.

22. Incubate elisa plate with 1:10,000 diluted EIA grade Goat anti-mouseIgG conjugated horseradish peroxidase in TBSW+0.5% ethanolamine, pH 7.0.Bring final volume to 150 ul/well. Incubate while shaking for thirtyminutes.

23. Wash plate as described in step 10.

24. Add 100 ul of ABTS/H202 solution to well. Incubate ten minutes whileshaking.

25. Add 100 ul of 0.2M HCL for 0.1M HCL final to stop thecolordevelopment reaction. Shake 1 minute at room temperature. Removebubbles with slow stream of air and read the ELISA plate in an ELISAplate reader at 410 nm.

6.2. Experimental Results

The results obtained for the tested compounds of the present inventionare set forth at Table 2.

                  TABLE 2                                                         ______________________________________                                        ELISA In Vitro Assay Results                                                  FLK-1R ELISA ASSAY RESULTS                                                    Compound  IC50(μM) Compound  IC50(μM)                                   ______________________________________                                        7         37.5        31        3.3                                           8         10.8        31        33.9                                          1         0.8         32        18.0                                          9         27.4        12        7.1                                           3         4.9         4         8.9                                           5         0.7         6         21.2                                          11        9.8         33        48.4                                          26        11          34        1.8                                           35        0.7         23        4.4                                           14        9.3         19        25.6                                          36        34.6        28        16.0                                          37        20.3        17        15.0                                          13        10.4        18        34.60                                         16        35.1        20        17.8                                          38        29.2        21        9.9                                           27        3.4         25        8.5                                           39        10          10        17.7                                          24        28.9        40        54.0                                          22        2.3         41        >50                                           42        >50         43        >50                                           44        >50         45        >50                                           46        >50         47        ˜50                                     48        >50         49        >50                                           50        >50         51        >50                                           52        >50         53        >50                                           54        ˜50   55        >50                                           56        >50         57        12.3                                          58        >50         59        >50                                           60        0.3         61        >50                                           22        2.3         62        >50                                           63        17.0        64        3.7                                           65        14.3                                                                ______________________________________                                    

7. Examples

Effect Of Compounds In In Vivo Studies

The ability of one of the compounds of the present invention,leflunomide (Compound 40), to inhibit ovarian, melanoma, prostate, lungand mammary tumor cell lines established as SC xenografts was examined.These studies were conducted using doses ranging from 12 to 20mg/kg/day.

7.1. Materials And Methods

The tumor cells were implanted subcutaneously into the indicated strainsof mice. Treatment was initiated on day 1 post implantation unlessotherwise indicated (e.g. treatment of the SCID mouse related to theA375 melanoma cell line began on Day 9). Eight (8) to ten (10) micecomprised each test group.

Specifically

Animals. Female athymic mice (BALB/c, nu/nu), BALB/c mice, Wistar ratsand Fisher 344 rats were obtained from Simonsen Laboratories (Gilroy,Calif.). Female A/I mice were obtained from Jackson Laboratory (BarHarbor, Me.). DA rats were obtained from B&K Universal, Inc. (Fremont,Calif.). Athymic R/Nu rats, DBA/2N mice, and BALB/c mice were obtainedfrom Harlan Sprague Dawley (Indianapolis, Ind.). Female C57BL/6 micewere obtained from Taconic (Germantown, N.Y.). All animals weremaintained under clean-room conditions in Micro-isolator cages withAlpha-dri bedding. They received sterile rodent chow and water adlibitum.

All procedures were conducted in accordance with the NIH Guide for theCare and Use Of Laboratory Animals.

Subcutaneous Xenograft Model. Cell lines were grown in appropriatemedium as described (See Section 6). Cells were harvested at or nearconfluency with 0.05% Trypsin-EDTA and pelleted at 450×g for 10 min.Pellets were resuspended in sterile PBS or media (without FBS) to asuitable concentration indicated in the Figure legends and the cellswere implanted into the hindflank of mice. Tumor growth was measuredover 3 to 6 weeks using venier calipers tumor volumes were calculated asa product of length x width x height unless otherwise indicated. Pvalues were calculated using the Students' t-test. su101 in 50-100 uLexcipient (dimethylsulfoxide, PBTE, PBTE6C:D5W, or PBTE:D5W) wasdelivered by IP injection at concentrations indicated in the Figurelegends.

Intracerebral Xenograft Model. For the mouse IC model, rat C6 gliomacells were harvested and suspended in sterile PBS at a concentration of2.5×10⁷ cells/ml and placed on ice. Cells were implanted into BALB/c,nu/nu mice in the following manner: the frontoparietal scalps of micewere shaved with animal clippers if necessary before swabbing with 70%ethanol. Animals were anesthetized with isofluorane and the needle wasinserted through the skull into the left hemisphere of the brain. Cellswere dispensed from Hamilton Gas-tight Syringes using 30 ga 1/2 inchneedles fitted with sleeves that allowed only a 3 mm penetration. Arepeater dispenser was used for accurate delivery of 4 uL of cellsuspension. Animals were monitored daily for well-being and weresacrificed when they had a weight loss of about 40% and/or showedneurological symptoms.

For the rat IC model, rats (Wistar, Sprague Dawley, Fisher 344, orathymic R/Nu; approximately 200 g) were anesthetized by an IP injectionof 100 mg/kg Ketaset (ketamine hydrochloride; Aveco, Fort Dodge, Iowa)and 5 mg/kg Rompun (xylazine, 2% solution; Bayer, Germany). After onsetof anesthesia, the scalp was shaved and the animal was oriented in astereotaxic apparatus (Stoelting, Wood Dale, Ill.). The skin at theincision site was cleaned 3 times with alternating swabs of 70% ethanoland 10% Poidone-Iodine. A median 1.0-1.5 cm incision was made in thescalp using a sterile surgical blade. The skin was detached slightly andpulled to the sides to expose the sutures on the skull surface. A dentaldrill (Stopiting, Wood Dale, Ill.) was used to make a small (1-2 mmdiameter) burrhole in the skull approximately 1 mm anterior and 2 mmlateral to the bregma. The cell suspension was drawn into a 50 uLHamilton syringe fitted with a 23 or 25 g a standard bevel needle. Thesyringe was oriented in the burrhole at the level of the arachnoidea andlowered until the tip of the needle was 3 mm deep into the brainstructure, where the cell suspension was slowly injected. After cellswere injected, the needle was left in the burrhole for 1-2 minutes toallow for complete delivery of the cells. The skull was cleaned and theskin was closed with 2 to 3 sutures. Animals were observed for recoveryfrom surgery and anesthesia. Throughout the experiment, animals wereobserved at least twice each day for development of symptoms associatedwith progression of intracerebral tumor. Animals displaying advancedsymptoms (leaning, loss of balance, dehydration, loss of appetite, lossof coordination, cessation of grooming activities, and/or significantweight loss) were humanely sacrificed and the organs and tissues ofinterest were resected.

Intraperitoneal Model. Cell lines were grown in the appropriate media.Cells were harvested and washed in sterile PBS or medium without FBS,resuspended to a suitable concentration, and injected into the IP cavityof mice of the appropriate strain. Mice were observed daily for theoccurrence of ascites formation. Individual animals were sacrificed whenthey presented with a weight gain of 40%, or when the IP tumor burdenbegan to cause undue stress and pain to the animal.

Immunohistochemistry. Acetone-fixed, 5 um frozen tissue sectionsuntreated xenograft tumors derived from human, rat, or murine tumorcells were analyzed by immunohistochemistry using highly specificreceptor antibodies. Briefly, non-specific binding sites were blockedwith 10% normal goat serum prior to the application of the primarilyantibody. Appropriate antibody concentrations were used to achieve thedesired sensitivity and specificity (rabbit anti-human PDGF-B receptor1:400, and affinity purified rabbit anti-mouse FLK-1 5.5 ug/ml). Tissuesections known to contain the protein of interest served as positivecontrols. Appropriate negative controls of normal rabbit IgG and mouseanti-chicken IgG of the same protein concentration and isotype as theprimary antibodies were used. The detection method was a three-stepindirect procedure and consisted of the primary antibody bound to abiotin labeled secondary antibody (goat anti-rabbit IgG 1:500) followedby streptavidin conjugated horseradish peroxidase.Diaminobenzidine/0.03%-hydrogen peroxide (1:200) (0.05w) was used as thechromogen/substrate. Tissue sections were counterstained withhematoxylin, dehydrated through ascending grades of ethanol, cleared inXylene Substitute, and coverslipped with Permount for microscopicevaluation. A "+" to "+++" grading system was used to identify theoverall intensity of the expression. One plus ("+") reflects lowintensity. Two pluses ("++") relates to medium intensity and threepluses ("+++") relates to high intensity. "T" is used in Table 4, below,to designate a tumor cell-specific staining reaction. "V" is used in theTable 4, below, to indicate a vascular endothelial cell-specificstaining reaction. "*" is used above to indicate that the analysis wascarried out using cytospins from the indicated cell lines. "NS" refersto "not significant" and "NT" refers to "not tested."

7.2. Experimental Results

Acetone-fixed frozen sections from xenograft tumors derived from humanor murine tumor cell lines were analyzed by immunohistochemistry (IHC),as discussed above, using highly specific receptor antibodies todetermine which tumor cell lines expressed the FLK-1 receptor.Specifically, the antibodies were obtained according to the procedureset forth at U.S. application Ser. No. 08/193,829. The results of theIHC analysis is set forth at Table 4:

                  TABLE 4                                                         ______________________________________                                        IHC Analysis of Tumors                                                        Tumor          FLK-1     % Inhibition                                         ______________________________________                                        C6             ++(V)     >95%                                                 SKOV3T         NT        >95%                                                 D1B            NT        95%                                                  SF763T         NT        85%                                                  U87MG          NT        75%                                                  L1210          NT        75%                                                  PC-3           +/++(V)   71%                                                  SF767T         NT        70%                                                  U118T          NT        57%                                                  Calu-6         ++/+++(V) 64%                                                  U373MG         NT        54%                                                  PA-1           ++(V)     53%                                                  A375           ++/+++(V) 53%                                                  A431           -*        NS                                                   MCF7           -         NS                                                   A549           NT        NS                                                   MCF7/HER2      -         NT                                                   SKOV3          NT        NT                                                   ______________________________________                                    

The in vivo experiments described above were then performed using thetumor cell lines which expressed FLK-1. The results were obtained in theabove-described in vivo experiments are set forth at Table 4.

                  TABLE 5                                                         ______________________________________                                        Effect Of Compound 40 On Tumor Growth In Vivo                                 Tumor   Cell             Dose   % Inhibi-                                     Type    Line    Strain   mg/kg/day                                                                            tion (day)                                                                            P <                                   ______________________________________                                        ovarian PA-1    nu/nu    20     53 (36) 0.04                                  melanoma                                                                              A375    nu/nu    20     53 (31) 0.03                                  melanoma                                                                              A375    SCID     15     53 (31) 0.002                                                 (day 9)                                                       prostate                                                                              PC-3    nu/nu    20     71 (45) 0.01                                  prostate                                                                              PC-3    SCID     12     47 (36) 0.001                                                 (day                                                                          15)                                                           lung    Calu-   nu/nu    20     64 (28) 0.0001                                        6                                                                     ______________________________________                                    

These studies show a significant inhibition of tumor growth inimmunocompetent animals treated with Compound 40. Specifically, as setforth above, Compound 40 effectively inhibited the growth of humanovarian (PA-1), human melanoma (A375), human prostate (PC-3), and humanlung (Calu-6).

The studies were repeated to test additional compounds, includingCompound 23. Compound 23 exhibited 41% inhibition at 20 mg/kg/day. Morespecifically, the results observed are set forth at Table 5.

                  TABLE 6                                                         ______________________________________                                        Effect Of Compounds In Vivo                                                   Compound        CALU-6 mg/kg/day                                              ______________________________________                                        4               no significant inhibition @ 20                                23              41% inhibition @20                                            12              no detectable inhibition @ 20                                 2               no detectable inhibition                                                      at 20 or 40                                                   40              63.9% inhibition @ 20                                         ______________________________________                                    

The apparent lack of inhibition for some of the tested compounds doesnot necessarily indicate lack of activity (inhibition of angiogenesisand/or vasculogenesis) and may be explained by, for example, thehalf-life of the compound tested in vivo or the dose administered in theexperiments.

8. Examples

In Vivo VEGF Pellet Model

In this model, VEGF is packaged into a time-release pellet and implantedsubcutaneously on the abdomen of nude mice to induce a `reddening`response and subsequent swelling around the pellet. Potential FLK-1inhibitors may then be implanted in methylcellulose near the VEGF pelletito determine whether such inhibitor may be used to inhibit the"reddening" response and subsequent swelling.

8.1. Materials And Methods

Materials. The following materials were used:

1) VEGF--human recombinant lyophilized product is commercially may beobtained from Peprotech, Inc., Princeton Business Park, G2; P.O. box275, Rocky Hill, N.J. 08553.

2) VEGF packaged into 21 day release pellets were obtained fromInnovative Research of America, using patented matrix driven deliverysystem. Pellets were packaged at 0.20, 0.21, or 2.1 μg VEGF/pellet.These doses approximate 10 and 100 ng/day release of VEGF. (InnovativeResearch of America, 3361 Executive Parkway, P.O. box 2746, Toledo, Ohio43606)

3) Methylcellulose

4) Water (sterile)

5) Methanol

6) Appropriate drugs/inhibitors

7) 10 cm culture plates

8) parafilm

Protocol. The following protocol was then followed to conduct the VEGFpellet model:

1) VEGF, purchased from Peprotech, was sent to Innovative Research forCustom Pellet preparation;

2) Methylcellulose prepared at 1.5% (w/v) in sterile water;

3) Drugs solublized in methanol (usual concentration range=10 to 20mg/ml);

4) Place sterile parafilm in sterile 10 cm plates;

5) 150 μl of drug in methanol added to 1.35 ml of 1.5% methylcelluloseand mixed/vortexed thoroughly;

6) 25 μl aliquots of homogenate placed on parafilm and dried into discs;

7) Mice (6-10 wk. Balb/C athymic nu/nu, female) were anesthetized viaisoflurane inhalation;

8) VEGF pellets and methylcellulose discs were implanted subcutaneouslyon the abdomen; and

9) Mice were scored at 24 hours and 48 hours for reddening and swellingresponse.

The specific experimental design used in this example was:

N=4 animals/group

Controls: VEGF pellet+drug placebo

VEGF placebo+drug pellet

8.2. Experimental Results

Compounds 5 and 15 were tested for activity according to theabove-described protocol. Both compounds inhibited the "reddening"response and subsequent swelling indicating an inhibition of bloodvessel formation and demonstrated activity against the FLK-1 receptor.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention, and any clones, DNA or amino acid sequences which arefunctionally equivalent are within the scope of the invention. Indeed,various modifications of the invention in addition to those describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are hereby incorporated by reference intheir entirety.

What is claimed:
 1. A method for treating diseases related tovasculogenesis and/or angiogenesis comprising administering to a subjecta therapeutically effective amount of a compound having the formula:##STR21## or a pharmaceutically acceptable salt thereof, wherein: R₁ andR₂ form a phenyl ring (CHCHCHCH),R₃ is H or formyl or chloro, and R₄ isphenyl, (3,4-dihydroxy)phenyl, (4-iodophenyl)amino,(3,4-dichlorophenyl)amino, (3-chlorophenyl)amino, (4-bromophenyl)aminoor n-propylamino.
 2. A method for inhibiting solid cell tumor growthcomprising administering to a subject a therapeutically effective amountof a compound having the formula: ##STR22## or a pharmaceuticallyacceptable salt thereof, wherein: R₁ and R₂ form a phenyl ring(CHCHCHCH),R₃ is H or formyl or chloro, and R₄ is (3,4-dihydroxy)phenyl,(4-iodophenyl)amino, (3,4-dichlorophenyl)amino, (3-chlorophenyl)amino.(4-bromophenyl)amino or n-propylamino.
 3. The method of claim 1 whereinsaid disease is selected from the group consisting of diabetes, diabeticretinopathy, hemangioma, glioma, melanoma, Kaposi's sarcoma, ovariancancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer,colon cancer and epidermoid cancer.
 4. A method for treating diseasesrelated to vasculogenesis and/or angiogenesis comprising administeringto a subject a therapeutically effective amount of the compound (E)-2-1-cyano-2-(5-bromo-3,4-dihydroxyphenyl)ethenylsulfonyl!-3-(3-bromo-4,5-dihydroxyphenyl)acrylonitrile, or a pharmaceutically acceptable salt thereof.